Project description:Background: Results obtained from our previous study using a label-free quantitation (LF) proteomic approach in which dynamic range compression was used to profile lower abundance proteins, demonstrated few proteins that were differentially abundant within the synovial fluid (SF) when comparing Autologous Chondrocyte Implantation (ACI) responders and non-responders at baseline (1). This study builds upon our previous findings by assessing higher abundance proteins within these SFs; providing a more global proteome analysis from which we can understand more fully the biology underlying ACI success or failure. Methods: Isobaric tagging for relative and absolute quantitation (iTRAQ) proteomics was used to assess SFs from ACI responders (mean Lysholm improvement of 33; n=14) and non-responders (mean Lysholm decrease of 14; n=13) at the two stages of surgery (cartilage harvest and chondrocyte implantation). Differentially abundant proteins were investigated using pathway analyses and the iTRAQ proteomic dataset was combined with our published proteomic dataset of dynamically compressed SFs, from which an interactome network model of systemic protein interactions was generated. Results: iTRAQ proteomics has confirmed our previous finding that there is a marked proteome shift in response to cartilage harvest (70 and 54 proteins demonstrating ≥2.0 fold change between Stages I and II in responders and non-responders, respectively) and has highlighted 28 proteins that were differentially abundant between responders and non-responders to ACI, that were not found in the LF study; 16 of which were altered at baseline. Two protein abundance changes (Complement C1S subcomponent and Matrix metalloproteinase 3 (MMP3), have been biochemically validated. Combination of the iTRAQ and LF proteomic datasets has generated in-depth SF proteome information that has been used to generate interactome networks representing ACI success or failure, from which functional pathways that are dysregulated in ACI non-responders have been identified. Conclusions: Several candidate biomarkers for baseline prediction of ACI outcome have been identified. A holistic overview of the SF proteome in responders and non-responders to ACI has been profiled providing a better understanding of the biological pathways underlying clinical outcome, particularly the differential response to cartilage harvest in non-responders.

Project description:Fibroblasts have only recently been identified as important effector cells in inflammation. In this study, human dermal fibroblasts were inflammatory stimulated with interleukin-1beta and comprehensively analysed with respect to proteins, eicosanoids and metabolites. For eicosadomics, we have established a data-dependent shotgun analysis method capable of identifying inflammation-regulated lipids of yet unknown function. Several classical inflammatory agonists were found induced, including PGA2, PGB2, PGE2 and TXB2, but also modulators such as PGA3 and PGE3, while 8-HETE and several HODE family members remained unaffected. Using targeted metabolomics, several acylcarnithins, phosphatitylcholins and sphingomyelins were found significantly downregulated. Proteome profiling with orbitrap-MS demonstrated the strong induction of several chemokines, metalloproteinases and other effector molecules. Treatment of stimulated fibroblasts with dexamethasone almost completely abrogated the formation of all inflammation-induced eicosanoids and restored levels of acylcarnithins back to normal. As expected, the secretion of IL-6, MMP1, MMP3, CXCL2 and CXCL3 was strongly down-regulated. However, instead of counter-regulating, dexamethasone further enhanced consequences of inflammatory stimulation with respect to CXCL1, CXCL6, complement C3 as well as sphingomyelins. Shotgun secretome data were confirmed by targeted analysis with triple-quadrupol-MS. These molecules have been described to be involved in chronic inflammation. In peripheral blood mononuclear cells, actually dexamethasone successfully downregulated the formation of all detectable inflammation mediators. The present data suggest that successful pharmacological abrogation of the formation of lipid inflammatory mediators in fibroblasts may not suffice to suppress the release of several other powerful inflammatory mediators which we thus understand to be capable of establishing chronic inflammation states.

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Human synovial fluid proteomics reveals potential early-diagnostic biomarkers for ankylosing spondylitis

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Proteomic analysis of human synovial fluid reveals potential diagnostic biomarkers for ankylosing spondylitis.

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